Identification of glycosyltransferases that synthesize pectin heteroglycan block polymers and a structural motif required for their activity

Pectin is a family of plant cell wall complex polysaccharides and proteoglycans comprised of the glycan domains homogalacturonan (HG), rhamnogalacturonan I (RG-I), and the substituted HGs rhamnogalacturonan II (RG-II), xylogalacturonan and apiogalacturonan. The number, type, and arrangement of the pectic domains in different pectic polymers are unknown, as is the identity of the enzymes that link the domains together. Here we show that two Arabidopsis -1,4-galacturonosyltransferases, GAUT13 and GAUT14, in addition to previously reported HG acceptor-dependent and HG de novo synthesis activities, initiate and elongate HG on the non-reducing end of RG-I, identifying enzymes that synthesize pectic RG-I:HG heteroglycans. While other catalytic GAUTs did not initiate HG on RG-I acceptors, they were able to elongate HG once initiated on RG-I by GAUT13 and GAUT14. We show that the three different GAUT14 acceptors (UDP-GalA, HG, and RG-I) compete for the same binding pocket, and amino acids required for GAUT14 catalytic activity were identified using in vitro enzyme assays, computational modeling, and site-directed mutagenesis. The transfer of the GAUT14 hypervariable 1 (HV1) motif (RTFASKLQSRS) was sufficient and necessary to transfer RG-I:HG extension activity to GAUT10, which otherwise does not have this activity, representing a novel functional glycosyltransferase domain swapping strategy. The presence of all three catalytic activities in a GAUT13/14 homolog from the non-vascular seedless plant Physcomitrium established the early evolution of GAUT13/14 trifunctionality. The identification of GAUT13 and GAUT14 as RG-I:HG extension catalysts provides targets for probing in planta structure and function of RG-I:HG heteropolymers.